P1654: COMPLEMENT ACTIVATION REGULATES PLATELET HOMEOSTASIS BY ARRESTING THE ANTI-INFLAMMATORY EFFECTS OF INTERLEUKIN-35 IN IMMUNE THROMBOCYTOPENIA
Authors: R.-Y. Gui, Y. Wu, Y.-X. Chen, F.-Q. Liu, Q.-Y. Qu, X.-J. Huang, X.-H. Zhang
Journal: HemaSphere (2022)

Background: Immune thrombocytopenia (ITP) is an acquired autoimmune haemorrhagic disease. Enhanced complement activation has long been considered one of the major pathogenesis of ITP. Bone marrow (BM) mesenchymal stem cells (MSCs) are multipotent stromal cells with multidifferentiation capacities, haematopoiesis support and immunomodulatory effects. Our previous study confirmed that enhanced complement activation could induce apoptosis and dysfunction of MSCs in ITP and alterations in the location of megakaryocytes (MKs) in the thrombopoietic niche in BM (Haematologica, 2019). The results strongly indicated that enhanced complement activation in the BM niche of ITP plays a role in the pathogenesis of ITP by influencing platelet formation. However, the roles of the complement system in the maturity and differentiation of MKs or in peripheral platelet destruction remain poorly understood. Aims: To explore the effects of complement activation on platelet homeostasis in ITP. Methods: Thirty consecutive patients with ITP and 10 healthy donors were enrolled in our study. C1q, C4d, C3b and C5b-9 on the surface of MSCs and in peripheral blood from ITP patients and healthy volunteers were detected by indirect enzyme linked immunosorbent assay (ELISA). MSCs were isolated from BM samples and cocultured with human naive conventional T (Tconv) cells in Transwell culture dishes. iTr35 subsets were measured by flow cytometry. Enzyme-linked immunosorbent assay kits were used for the quantification of IL-35. The key transcription factors of T cell differentiation were detected by real-time quantitative-PCR analysis. Results: Sixteen ITP patients were assigned to the group with sublytic C5b-9 assembly on MSC membranes (MSC-ITP-C+ group), while the remaining 14 patients were assigned to the group without complement activation (MSC-ITP-C- group). The MSC-ITP-C+ group showed fewer iTr35 subsets (P<0.05) in BM than the MSC-ITP-C- group and healthy donors (MSC-Control group). Coculture with MSCs-ITP-C+ and Tconv cells resulted in a relatively lower number of iTr35 subsets and lower IL-35 levels. The addition of prostaglandin E2 restored Tconv cell proliferation in a dose-dependent manner and suppressed differentiation. Peripheral blood CD34+ progenitors from the MK-ITP group and MK-Control group were differentiated into megakaryocytes in vitro. The IL-35 receptors gp130 and IL-12Rβ were found to be expressed on human MKs, suggesting a role for IL-35 during megakaryopoiesis. When blood CD34+ cells were cultured for 10 days, the gp130 level in the MK-ITP group was significantly lower than that in the MK-Control group (P<0.0001), and the IL-12Rbeta level in the MK-Control group was significantly higher than that in the MK-ITP group (P<0.0001). Decreased CD41+ cells and CFU-MKs were also observed in the MK-ITP group. In vitro, the effect of IL-35 on CD34+ progenitor cells (MK-ITP+IL-35 group) was measured. Compared to the MK-ITP group, gp130 was higher on Day 10 in the MK-ITP+IL-35 group (P=0.017). The addition of IL-35 increased the total number of viable cells and CD41+ cells by approximately 10% on average through the phosphorylation of STAT1 and STAT3. Similar effects were also observed in ITP mice. Summary/Conclusion: Sublytic C5b-9 deposition on MSCs displayed a defective role in regulating iTr35 subsets, resulting in the downregulation of IL-35 in ITP. Defective megakaryopathies in ITP were also correlated with the downregulation of the IL-35 receptor. This study sheds light on a novel mechanism of ITP pathogenesis and provides potential therapeutic targets in ITP patients.